Depression is a chronic and recurrent disorder, associated with high morbidity and risk of suicide. Leptin was firstly described as an anti-obesity hormone, but several actions of leptin in CNS have been reported. In fact, leptin regulates dopaminergic neurotransmission in mesolimbic areas and has antidepressant-like properties in stress-based models. In the present study, we investigated, for the first time, putative antidepressant-like effects of leptin in an animal model of depressive-like behaviors induced by lipopolysaccharide (LPS), and the potential involvement of dopamine receptors as mediators of those behavioral effects. Mice were injected leptin  or imipramine prior to LPS administration. To evaluate the involvement of dopamine receptors, different experimental groups were pretreated with either the dopaminergic antagonist SCH23390, for D1 receptors or raclopride, for D2/D3 receptors, prior to leptin injection. Twenty-four hours post-LPS, mice were submitted to the forced swimming and sucrose preference tests. In addition, IL-1β levels were determined in the prefrontal cortex (PFC), hippocampus and striatum. BDNF levels were measured in the hippocampus. Our results showed that leptin, similarly to imipramine, prevented the core behavioral alterations induced by LPS (despair-like behavior and anhedonia), without altering locomotion. In neurochemical analysis, leptin restored LPS-induced changes in IL-1β levels in the PFC and striatum, and increased BDNF levels in the hippocampus. The blockade of dopamine D1 and D2/D3 receptors inhibited leptin's antidepressant-like effects, whilst only the blockade of D1-like receptors blunted leptin-induced increments in prefrontal IL-1β levels. Our results indicate that leptin has antidepressant-like effects in an inflammatory model of depression with the contribution, at least partial, of dopamine receptors. Depression is the third leading source of years lived with disability worldwide and has a lifetime prevalence of 14.6% among people living in high-income countries . In spitAccumulating evidence indicates that peripheral immune activation and neuroinflammation may contribute to the development of depression , 6. For Several lines of evidence also point to the involvement of Gram-negative bacteria in the pathophysiology of depression. For example, depression accompanied by high plasma IgA responses to antigens/lipopolysaccharide (LPS) of commensal gut bacteria and increased root canal LPS in patients with comorbid depression and chronic apical periodontitis , 15. MorIndeed, the animal model of depressive-like behaviors induced by the systemic administration of LPS was first proposed by Yirmiya . EarlierAbnormalities in the reward brain circuit have been well-described in depressive patients as well as in several animal models of depression , 29. DepNeuroinflammation triggers alterations in dopamine brain circuits. In this regard, depressive behavior following interferon (IFN)-α administration to rhesus monkeys was related to lower cerebrospinal fluid (CSF) concentrations of the dopamine metabolites, homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) . Indeed,Besides regulating motivated behavior, consistent evidence suggests that mesolimbic dopaminergic system also influences important aspects of feeding behavior and energy expenditure . Indeed,The role of leptin in depression has been extensively investigated. Some studies have demonstrated that chronic stress paradigms impair serum leptin levels, and the deletion of its receptor (long form) in hippocampus and VTA induces depressive- and anxiety-like behaviors , 42. AlsDespite the well-known effects of leptin on dopamine signaling, to our knowledge, the involvement of dopamine receptors on leptin-induced antidepressant-like effects remain unexplored. Therefore, based on the putative role of dopaminergic signaling in the central effects of leptin and the absence of studies exploring the effects of this hormone in animal model of depressive-like behavior induced by immune challenge, we decided to test the hypothesis that leptin could prevent LPS-induced depressive-like behavior in mice and that this effect is mediated, at least partly, by the participation of D1- and/or D2-like dopamine receptors. Furthermore, we assessed whether leptin's antidepressant-like effects could be associated with alterations in IL-1β levels in limbic brain areas  and/or hippocampal BDNF levels in the brain of mice challenged with LPS.ad libitum. All experimental procedures were performed between 8 a.m. and 2 p.m., according to the Guide for the Care and Use of Laboratory Animals, from the US Department of Health and Human Services  obtained from the Animal House of the Universidade Federal do Ceará. Animals were housed 8 per cage under standard animal housing conditions, polycarbonate cages (42 × 20.5 × 20 cm), temperature 22 ± 1°C, humidity 60 ± 5%, 12 h light-dark cycle (light on 6 a.m.) and food/water Services  and to tEscherichia coli, strain 055:B5 and R(+)-SCH23390 hydrochloride were obtained from Sigma-Aldrich Corp., St. Louis, USA. Recombinant Mouse Leptin Protein was purchased from R & D Systems. Raclopride was obtained from Tocris Bioscience, Bristol, United Kingdom. The drugs were administered at the following doses (mg/kg): LPS 0.5, leptin 1.5, imipramine 10, SCH23390 0.015 and raclopride 0.04. All drugs were freshly prepared and diluted in saline solution. All other chemicals used were of analytical grade.Lipopolysaccharide (LPS) from The first protocol of the study consisted of behavioral determinations to test the antidepressant-like effect of leptin in LPS acute model . To do tThe second protocol was designed to determine the involvement of dopamine receptors in the antidepressant-like effects of leptin, by a systemic blockage of D1-like or D2-D3 receptors . In thisOpen field, forced swimming or sucrose preference tests were conducted to assess behavioral alterations. For these tests the animals were assigned into two distinct sets. The first set of mice comprised 6–10 animals/group, which were tested in the open field and forced swimming tests, in this order. The second set of animals was used for sucrose preference test comprising 6 animals/group. Distinct groups of animals were treated with the drugs and sacrificed for neurochemical determinations. The brain areas prefrontal cortex (PFC), hippocampus and striatum were dissected and immediately stored at −80°C until assayed. As hippocampus is one the main area that maintain active neurogenesis in adult brain, a mechanism involved in the response to antidepressants , we deciThe primary outcome of this study was to determine changes in locomotion, behavioral despair (by the forced swimming test) and anhedonia (by the sucrose preference test) in animals administered leptin as well as the participation of dopamine receptors. The secondary outcome was to verify alterations in inflammatory cytokines and BDNF in brain areas related to mood regulation.In this test, animals were subjected individually to the analysis of the depressive-like behavior, based on a model adapted from Porsolt et al. . In thisThis test was adapted from the model initially proposed by Archer  and was The test was performed 24 h after LPS administration to evaluate anhedonia. The procedure consisted of an adaptation period 72 h before the test in which mice were trained to adapt to sucrose solution with two bottles of 1% (w/v) sucrose solution placed in each cage. Twenty-four hours later, sucrose solution in one bottle was replaced with tap water during 24 h. After this adaptation period, mice were deprived of water and food for further 24 h. For the sucrose preference test mice were housed in individual cages with free access to two bottles containing 100 ml of sucrose solution (1% w/v) and 100 ml of water. After 1 h, the volumes of consumed sucrose solution and water were recorded and the sucrose preference was calculated as follows: % sucrose consumption = sucrose consumption/(water + sucrose consumption) × 100 .Each brain area was homogenized in 8 volumes of PBS buffer with protease (EMD Biosciences) and phosphatase (Sigma-Aldrich) inhibitors and centrifuged . For IL-1β determinations 50 μL samples were used. The immunoenzymatic assay (ELISA) was performed according to the manufacturer's protocol  and expressed in pg/g tissue.Each hippocampus was homogenized in 20 volumes of PBS buffer with protease (EMD Biosciences) and phosphatase (Sigma-Aldrich) inhibitors and centrifuged . For BDNF determinations 50 μL samples were used. The immunoenzymatic assay (ELISA) was performed according to the manufacturer's protocol ELISA  and expressed in pg/g tissue.post-hoc test. In the case of two-way ANOVA, the factors “LPS model”  and “drug treatment”  were used. The significance level was set at P ≤ 0.05. GraphPad Prism 6.0 Version for Windows, GraphPad Software  was used.Data from behavioral and neurochemical determinations are present as mean ± S.E.M. (standard errors of the mean). Kolmogorov–Smirnov normality test was conducted to verify the normal distribution of data. Statistical analyses were conducted by regular one- or two-way ANOVA with Tukey's multiple comparisons as F = 19.61, P < 0.0001], with significant main effect of “drug treatment” . As expected, LPS alone significantly increased immobility time (P < 0.0001) when compared to control animals. Pretreatment with leptin kept immobility time akin to control animals . On the other hand, imipramine decreased mice immobility time in relation to control group when administered alone (P = 0.0070) and when administered before LPS (Imi+LPS group) in relation to LPS-challenged group (P < 0.0001).As a first step, we evaluated the antidepressant-like effect of leptin and of the standard antidepressant imipramine in the forced swimming test . Two-wayP < 0.0001; Rac+Lep+LPS vs. Lep+LPS, P = 0.0002). On the other hand, neither D1-like nor D2/D3 receptor antagonists prevented the reduction in immobility time observed in Imi+LPS group in relation to LPS group .In the evaluation of forced swimming in animals submitted to the second protocol , pre-admF = 0.4668, P = 0.6307] (P = 0.0038). Still in the open field test, the measure of anxiety-like behavior evaluated by the time spent in the center of the field  = 14.22, P = 0.0005]. We observed that LPS-treated and Lep+LPS mice presented a decrease in the time spent in the center of the field in relation to saline-treated mice (P < 0.05).We next determined alterations in the locomotor activity to better address LPS-induced sickness behavior in these mice, a complex behavioral phenotype associated with hypolocomotion . In our  0.6307] . This re 0.6307] , we onlyhe field  revealedF = 7.729, P = 0.0020], with significant main effect of “LPS model” . The results showed that 24 h after the endotoxin administration, mice displayed a significant decrease in sucrose preference when compared to control group (P < 0.0001). The administration of leptin (P = 0.0043) or imipramine (P = 0.0004) prevented the decrease in sucrose preference induced by LPS.To better evaluate LPS-induced anhedonia, sucrose preference test was performed . In FiguP = 0.0026) or Rac+Lep+LPS (P < 0.0001) was lower than the observed in the control group, being like LPS group, revealing that both D1-like and D2/D3 antagonism abrogate the effect of leptin in this test. Imipramine (P = 0.0004), as leptin (P = 0.0039), prevented the decrease in sucrose preference in LPS-treated animals, although pretreatment with D1 and D2/D3 receptor antagonists did not change the effects of imipramine in sucrose consumption .In the second protocol , the perP = 0.0287)  and striatum (P = 0.038) . Pretreatment with imipramine (P < 0.0001) or leptin (P < 0.001) significantly decreased IL-1β levels in these brain areas when compared to LPS group. The antagonism of D1-like receptors partially inhibited the effects of leptin in PFC IL-1β levels. We did not observe a significant difference in this parameter in the group SCH+Lep+LPS in relation to Sal+LPS or control groups. No significant difference was observed in the evaluation of IL-1β levels in the hippocampus.Since LPS-induced depressive-like behavior is related to the induction of pro-inflammatory cytokines in the brain, the levels of IL-1β were measured . We founP = 0.0178). The group Lep+LPS also presented increased levels of BDNF when compared to control (P = 0.0003), while in the group pretreated with imipramine there was no significant alteration. The groups treated with SCH+Lep+LPS (P < 0.0001) or Rac+Lep+LPS (P = 0.0005) maintained the increase in BDNF levels observed in Lep+LPS treated mice being the results significant in relation to saline group (P < 0.05). In the case of SCH+Lep+LPS group, the increase in BDNF levels was also significant in relation to Sal+LPS group (P = 0.0041) .Finally, because oxidative stress and inflammation alters the brain levels of neurotrophic factors, BDNF content was evaluated in the hippocampus , the maiIn the present study, we are showing the antidepressant-like effects of leptin in LPS-induced inflammatory model of depression through two established behavioral paradigms, namely forced swimming and sucrose preference test. We also, demonstrated the involvement of D1-like and D2/D3 dopamine receptors in the behavioral effects of leptin, as well as in the ability of this hormone to rescue LPS-induced increases in IL-1β levels in the PFC and striatum. Interestingly, D1-like receptors blockade blunted the effects of leptin on IL-1β levels in the PFC, but not in the striatum. Additionally, hippocampal BDNF levels were increased in leptin-treated mice and unaffected by the blockade of dopamine receptors. Regarding anxiety-like behavior evaluated in the open field test, leptin was not able to prevent LPS-induced anxiety-like behavior. Therefore, this study brings new evidence about the antidepressant-like properties of leptin, while suggests the participation of dopaminergic receptors in the behavioral effects of this hormone as well as the participation of D1-like receptors in its anti-inflammatory effect in the PFC.Leptin's antidepressant-like effect was already demonstrated in stress models of depression, such as chronic unpredictable stress and chronic social defeat. Indeed, this previous study showed that animals exposed to stress-induced depression present low plasma levels of leptin, while leptin treatment restores these parameters . These aRegarding LPS-induced depressive-like behavior, it is well known that animals injected with LPS develop an acute mild state of nosothymia, termed sickness-behavior. However, 24 h post LPS, a state of depressive-like behavior emerges, a time-point when motor activity and food and drink consumption return to normal , 22. AccFurthermore, by using the number of entries in the center of the open field as a parameter of anxiety-like behavior, we observed that LPS-induced anxiety-like behavior was not prevented by leptin administration. This absence of anxiolytic effect by leptin in this inflammatory model of depression must be confirmed in future studies. In a previous study of our group, we did not observe anxiety-like alterations in the plus maze test 24 h post LPS, only 1.5 h after the endotoxin challenge, a time-point related to sickness behavior .A number of studies advocate for abnormalities in dopaminergic neurotransmission in major depressive disorder . InitialSome studies have reported that immune-inflammatory stimuli, such as LPS, or direct cytokine administration can severely affect dopaminergic system to mediate depressive symptoms, such as anhedonia and psychomotor retardation . In factAs aforementioned, neuroinflammatory alterations observed in depression and modeled by inflammatory models of depression can compromise dopamine neurotransmission in brain areas related to motivation, such as striatum, and to the integration of motivation and cognitive control for decision making, such as PFC . NoteworLeptin seems to regulate brain-reward circuit mainly by altering dopamine signaling. Evidences for leptin regulation of reward circuit are the reversal of drug-seeking behavior induced by heroin withdrawal  and incrIn the present study, we are showing the participation of D1-like and D2/D3 dopamine receptors in leptin antidepressant-like action in LPS-induced depressive-like behavior. Despite evidence indicating the involvement of dopaminergic neurotransmission in leptin effects, no previous study had evaluated the participation of dopamine receptors in the antidepressant mechanism of this hormone. Indeed, strong evidences advocates for the participation of dopamine receptors in depression: (i) chronic treatment with antidepressants of different classes increase the striatal expression of dopamine D2/D3  and D1-DIn our study, the blockade of D1- and D2-like dopamine receptors equally abrogated leptin effects in the forced swimming and sucrose preference tests. It is worth to mention that leptin regulatory effect in hedonic feeding is dependent on dopamine signaling through D2 receptor activation, and is inhibited by the pharmacological and genetic deletion of this receptor . Also, lRegarding IL-1β levels, our results point to a possible anti-inflammatory mechanism mediating leptin antidepressant-like effect. In this study, we assessed IL-1β levels as a parameter of active inflammation in relevant brain areas related to mood control. Indeed, leptin plays an important regulatory role in the immune system, in addition to its already established neuroendocrine functions. The levels of leptin seem to rapidly increase in acute inflammatory conditions, such as cholecystectomy and sepsis, particularly favored by cytokines such as TNF-α, IL-6, and IL-1β .Nevertheless, it was also reported that leptin exhibits some peripheral anti-inflammatory properties . In the According to our results, leptin plays an apparent anti-inflammatory effect as it reduced IL-1β levels in the PFC and striatum of mice challenged with LPS. It is important to note, that in our experimental conditions, we tested leptin effects in an established pro-inflammatory milieu induced by LPS, but not in control conditions, as performed in the abovementioned studies. Additionally, a recent study demonstrated leptin's anti-inflammatory actions in primary glial cell cultures challenged by the pro-inflammatory cytokines TNFα, IL-1β, and IFN-γ .Our results also pointed to an interesting involvement of D1-like receptors in leptin anti-inflammatory effect in the PFC. Previous evidence showed that the activation of dopamine receptors can modulate cytokine production in immune cells . In thisNeurotrophic factors are critical regulators of the formation and plasticity of neuronal networks. BDNF is an abundant neurotrophin in the brain involved in neuronal proliferation and differentiation . ExperimIn our experimental conditions, 24 h post LPS, we did not observe decreases in hippocampal BDNF levels, although pretreatment with both imipramine and leptin increased this parameter compared to saline and LPS-treated animals. This absence of alteration in hippocampal BDNF levels 24 h post LPS was also observed in previous studies of our research group .This variability in BDNF 24 h post LPS, observed in our results, might be explained by the release of the immature form of BDNF, namely proBDNF, which has apoptotic effects. Regarding leptin effects, a previous study reported that the subcutaneous administration of this peptide increases BDNF levels in the hippocampus of non-obese mice and simultaneously induces an antidepressant-like effect in forced swimming test . BesidesThe present study has some limitations. Indeed, human depression is a syndrome, comprising symptoms such as depressed mood, diminished interest or pleasure, fatigue or loss of energy, social withdrawal and cognitive dysfunction, among others, and for this reason should be evaluated in experimental models by behavioral tests related to these symptoms. In the present study, we focused on core alterations of depression, namely behavioral despair and anhedonia and in a less extent anxiety-like alterations. Hence, it is important to address, in future studies, leptin's effects in other behavioral tests related to anxiety, social interaction, and cognitive function. Furthermore, it is important to determine alterations in dopaminergic receptors in brain areas of mice exposed to LPS depression model, to better address the participation of these receptors in this inflammatory model as well as the effects of leptin administration in these receptors.In summary, the present study demonstrated leptin's ability to rescue LPS-induced depressive-like behaviors including anhedonia and despair-like behavior, and some immune changes, namely increases in IL-1β levels in the PFC and striatum. This is the first study showing the involvement of dopamine receptors in the antidepressant-like effects of leptin, suggesting the participation of D1-like receptors in this hormone immunoregulatory effects in the PFC. Of note, a previous study have pointed to hippocampus as the main site of leptin antidepressant effects . TherefoRC conceived the study, conducted the experiments, and wrote the first draft. AF, VT, CM, AQ, and NG conducted the experiments, and analyzed and interpreted the data. MM analyzed the data, interpreted the data, and provided intellectual input to the manuscript. DM and AC conceived the study, supervised the experiments, analyzed and interpreted the data, and provided intellectual input to the manuscript. All authors have reviewed and approved the manuscript prior to submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pulmonary regurgitation is a common complication after tetralogy of Fallot repair, resulting in right ventricular dysfunction, arrhythmia, and sudden death. However, the indications and optimal timing for pulmonary valve replacement are not fully known. We describe a case in which a four-dimensional imaging tool was useful in the decision to re-operate, thus resulting in decreased energy loss and improved right ventricular function after the re-operation for tetralogy of Fallot.2, and the cardiac index was 1.78 L/minute per m2. Flow energy loss was 2.93 mW, which is estimated to be three times higher than normal controls. An electrophysiological study showed an intact anterior internodal pathway and a slow pathway just through the outside of the right atriotomy line scar, which is supposed to cause a re-entry circuit. We decided to perform a pulmonary valve replacement and a right maze procedure. A 27 mm bioprosthetic valve was implanted in the native pulmonary annulus with a supra-annular position. Concomitantly, the right maze procedure was performed. A four-dimensional flow magnetic resonance imaging done 3 months later showed that right ventricular end-diastolic/end-systolic volume index had significantly reduced to 85.24/55.41 mL/m2 and the cardiac index had increased from 1.78 to 2.58 L/minute per m2. Energy loss had greatly improved from 2.93 to 1.48 mW.A 54-year-old Japanese woman visited our hospital due to palpitations and wide QRS tachycardia with persistent tiredness for several months. She underwent repair of tetralogy of Fallot when she was 2-years old. An electrocardiogram showed prolonged QRS duration (199 msec) with a complete right bundle branch block and an echocardiograph demonstrated that her right ventricle was highly enlarged and had poor contraction, and severe pulmonary valve regurgitation with one leaflet flail. Four-dimensional flow magnetic resonance imaging demonstrated that regurgitant volumes and regurgitant fractions of pulmonary regurgitation were calculated as 63.12 ml and 54.0%, respectively. Right ventricular end-diastolic/end-systolic volume index was 169.54/99.76 mL/mA four-dimensional imaging tool was useful in the decision to re-operate, thus resulting in decreased energy loss and improved right ventricular function after the re-operation for tetralogy of Fallot.The online version of this article (10.1186/s13256-018-1964-9) contains supplementary material, which is available to authorized users. Survival after tetralogy of Fallot (TOF) repair has been improving, but long-term mortality is still unsatisfactory, especially 25 years or more after the repair . PulmonaWe describe a case in which a four-dimensional imaging tool was useful in the decision to re-operate, thus resulting in decreased energy loss and improved RV function after the re-operation for TOF.A 54-year-old Japanese woman visited our hospital due to palpitations and wide QRS tachycardia with persistent tiredness for several months. She underwent repair of TOF when she was 2-years old. After the repair, no follow-up check was performed. During the period, she gave birth to three children. She underwent atrial flutter ablation (cavo-tricuspid isthmus block line) when she was 50-years old and 51-years old.On admission, she had wide QRS tachycardia of 180 beats/minute, but it spontaneously converted to sinus rhythm. This paroxysmal wide QRS tachyarrhythmia of a few minutes’ duration was easily observed several times.2, and the cardiac index (CI) was 1.78 L/minute per m2. Flow energy loss (FEL) calculated from four-dimensional flow MRI was 2.93 mW, which is estimated to be three times higher than normal controls  with a complete right bundle branch block and an echocardiograph demonstrated that her right ventricle was highly enlarged and had poor contraction, and severe pulmonary valve regurgitation with one leaflet flail Additional file 2:Movie S2. Flow Energy Loss before PVR. (MP4 1096 kb)We decided to perform a PVR and the right maze procedure because the energy loss of the right side of her heart system was high and, additionally, arrhythmic therapy was needed, even if the extremely dilated RV size did not get restored. Cardiopulmonary bypass (CPB) was instituted with the ascending aorta and bicaval cannulation. Anterior and posterior pulmonary valve leaflets were prolapsed and the left leaflet was highly degenerated. The pulmonary annulus was intact with sufficient size; thus, a bioprosthetic valve was implanted in the native pulmonary annulus with a supra-annular position. After all the leaflets were resected, a Mosaic Ultra® 27 mm  was placed on the supra-annular position. Concomitantly, the right maze procedure was performed. The right atrium was plicated with the removal of the scar, adding the incision to the lateral side of the atrium, and cryoablation on the posterior and anterior walls of the atrium from the incision line to the tricuspid isthmus and to the inferior vena cava.2 and her CI had increased from 1.78 to 2.58 L/minute per m2. Energy loss had greatly improved from 2.93 to 1.48 mW  , and thaArrhythmias are also a common late complication of repaired TOF. Atrial arrhythmias may be present in 65% of patients after TOF repair . Atrial A four-dimensional imaging tool was useful in the decision to re-operate, thus resulting in decreased energy loss and improved RV function after the re-operation for TOF.


N-Acetylhexosamine oligosaccharides terminated with GalNAc act as selective ligands of galectin-3, a biomedically important human lectin. Their synthesis can be accomplished by β-N-acetylhexosaminidases (EC 3.2.1.52). Advantageously, these enzymes tolerate the presence of functional groups in the substrate molecule, such as the thiourea linker useful for covalent conjugation of glycans to a multivalent carrier, affording glyconjugates. β-N-Acetylhexosaminidases exhibit activity towards both N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine  moieties. A point mutation of active-site amino acid Tyr into other amino acid residues, especially Phe, His, and Asn, has previously been shown to strongly suppress the hydrolytic activity of β-N-acetylhexosaminidases, creating enzymatic synthetic engines. In the present work, we demonstrate that Tyr470 is an important mutation hotspot for altering the ratio of GlcNAcase/GalNAcase activity, resulting in mutant enzymes with varying affinity to GlcNAc/GalNAc substrates. The enzyme selectivity may additionally be manipulated by altering the reaction medium upon changing pH or adding selected organic co-solvents. As a result, we are able to fine-tune the β-N-acetylhexosaminidase affinity and selectivity, resulting in a high-yield production of the functionalized GalNAcβ4GlcNAc disaccharide, a selective ligand of galectin-3. N-Acetylhexosamine oligosaccharides are attractive synthetic targets since they act as potent ligands of biotechnologically or biomedically attractive lectins [N-Acetylhexosaminidases (EC 3.2.1.52) have the advantage of tolerating a number of functional groups incorporated in both the donor and acceptor carbohydrate molecule [ lectins ,2. β-N-Adases EC .2.1.52 hmolecule ,5. Besidmolecule ,7. Suitamolecule ,9.N-acetylhexosaminidases and favorably shift the reaction equilibrium towards the synthetic mode [N-acetylhexosaminidases—the ratio of activities towards N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine  substrates. In most wild-type enzymes, this ratio is relatively undistinctive, ranging between ca 0.7–1.5 [N-acetylhexosaminidase from Talaromyces flavus (TfHex), recombinantly expressed in Pichia pastoris [We have recently discovered that a point mutation of the active-site amino acid Tyr to other amino acid residues, especially Phe, His, and Asn, can strongly suppress the hydrolytic activity of β-tic mode . In this 0.7–1.5 , though  0.7–1.5 . Moreovepastoris . This enpastoris . By combN-acetylhexosaminidases.GalNAc-terminated oligosaccharide epitopes rarely occur in mammalian glycoproteins ,16 or paTfHex—not only for its synthetic capability but especially for the exceptional stabilization of its GalNAcase activity against organic solvents during the synthetic procedure. These features greatly enhanced the yield of preparative GalNAcylations and enabled a large-scale preparation of the desired LacdiNAc disaccharide, which may further be applied for multivalent presentation in galectin-3-binding glycomimetics. The altered substrate specificity of TfHex upon mutating the Tyr470 residue to Phe, His, or Asn was thoroughly studied by molecular modeling and substrate docking. The present work demonstrates the critical importance of the Tyr470Phe mutation in TfHex, namely Tyr470Phe, Tyr470His, and Tyr470Asn, were cloned in the pPICZαA vector as described previously [P. pastoris KM71H. For the preparative enzyme production, the respective enzymes were secreted into the minimal cultivation medium upon induction by methanol. The mutant variants were purified by cation-exchange chromatography at pH 3.5 in a single purification step, which yielded the proteins in a high purity for further biochemical characterization . The specific activities given in The genes of three Tyr470 mutants of eviously , and traTfHex variants tested, GalNAcase activity was at its peak at a slightly acidic pH (4–5) and it declined at pH approaching either lower or higher values. Thus, for example at pH 7, the ratio of GalNAcase/GlcNAcase declined approximately twice compared to pH 5 in the WT TfHex. At pH higher than 8, all enzyme variants were substantially inactivated. The by far highest GalNAcase activity at changing pH was found in the Tyr470Phe mutant. There, quite a stable ratio of GalNAcase/GlcNAcase activity was observed over a large span of pH 2.5–8.5. This finding clearly shows the importance of Tyr470Phe mutation for stabilizing and protecting the GalNAcase activity in TfHex. The pH optima profiles of both GalNAcase and GlcNAcase activities of all the enzyme variants as well as of the wild type were determined as shown in Km, kcat) of the hydrolytic reactions with pNP-GlcNAc and pNP-GalNAc substrates with all enzyme variants were measured and compared to the wild type . This trend is also well visible on the values of Km. On the other hand, in both the His and Asn mutants, the GalNAcase activity is strongly suppressed compared to the GlcNAcase activity, which clearly reflects on the ratios of respective catalytic efficiencies . This considerable decline is caused by reduced affinity of the pNP-GalNAc substrate to the enzyme active site as reflected on the increased values of Km. These structure activity relationship conclusions were supported by in silico substrate docking and molecular modeling as shown further. Naturally, since the present mutant variants have a strongly suppressed hydrolytic activity, the hydrolytic catalytic efficiency is lower compared to the WT. It follows from the kinetic parameters that this behavior is caused mainly by reduction of TfHex mutant variants and molecular dynamics simulations, the previously described homology model of TfHex [pNP-GlcNAc and pNP-GalNAc docked in the active site of WT and of mutant variants of TfHex are shown in pNP-GlcNAc and Tyr470Phe-pNP-GalNAc. In contrast, the highest binding energies were observed for both substrates in the Tyr470Asn mutant, which agrees well with the higher Km values  vary between 6.76 and 7.4. Though a better binding score was found for the neutral His form , and consequently, in the increase in Km value. β-ractions . One of ractions . DynamicpNP-GalNAc, does not require a large reorientation of active site amino acids and features low RMSF values. Most of the studied residues are very stable over the equilibrated period of the molecular dynamics run (2–10 ns). The most flexible residues are Glu332 and Trp509, which are responsible for aglycon binding. The orientation of Trp509 is stabilized by a hydrogen bond network between its amide hydrogen, the C-6 hydroxyl of substrate, and the carboxylic oxygen of Asp472 [TfHex probably results from the general stability of the active site in this mutant variant, especially when pNP-GalNAc is bound. Moreover, the presence of Phe may bring additional aromatic π-stacking with the Trp ring. In contrast, in the Tyr470Asn variant, Asp472 changes its orientation more significantly than in the other mutants and WT.The flexibility of enzyme active site amino acids required to accommodate the substrate is characterized by the root-mean-square fluctuation of atomic positions (RMSF) of enzyme active-site residues . In the f Asp472 . The mutpNP-GlcNAc and pNP-GalNAc docked into the active site of mutant variants of TfHex are shown in The substrates N-acetyl group. The p-nitrophenyl group of pNP-GlcNAc is rotated with respect to Trp509 position in WT  led to the increased distance between the catalytic residue Glu371 and pNP-GalNAc  may contribute to the mutant preference for pNP-GlcNAc substrate. In the complex with the Tyr470Asn mutant c,f, wateTfHex was identified as the most promising candidate for a successful GalNAc-ylation reaction. Therefore, we decided to study the influence of various reaction media containing organic water-miscible aprotic co-solvents on the GalNAcase/GlcNAcase activity ratio of this mutant and compare it to the WT. Four solvents were selected for this aim: acetonitrile, acetone, t-butanol, and DMSO. The results are shown in From the biochemical characterization as well as from the docking experiments, the Tyr470Phe mutant variant of v/v DMSO). Astonishingly, the mutant enzyme kept a good activity even at high solvent concentrations . This is quite a different case from what was observed in the WT TfHex  epitope  and the reaction may further be scaled up. The thiourea linker may be deprotected under acidic conditions [The utility of the Tyr470Phe itope 3; . This dinditions  and readnditions  or otherpNP-GlcNAc) can be transferred to another substrate . Naturally, there are limits as to what results may be used for practical application in the upscaled synthetic reactions. For example, manipulation of reaction pH may not be practically applicable to a large extent, since many glycoside and oligosaccharides are relatively sensitive towards extreme pH values. Similarly, a considerable shift of the temperature of a synthetic reaction is hardly recommendable in the case of glycosidases. This is because higher temperatures generally lead to a lower transglycosylation yield in favor of hydrolysis, which is rather counterproductive when we aim at oligosaccharide synthesis. Nevertheless, a considerate choice of optimum reaction conditions may bring great benefits for reaction yields.It clearly follows from the present study that the impact of pH value, the presence of co-solvents, and other means of reaction engineering are strictly dependent on a particular enzyme-substrate combination and any general rules may hardly be applied. Thus, medium engineering must be performed with the particular substrate in question, and strictly speaking, no conclusions found for one substrate , compared with UDP-GalNAc-10 mg for 315 USD or UDP-GlcNAc-250 mg for 415 USD. Notably, though less expensive, UDP-GlcNAc requires a tandem reaction with UDP-GlcNAc-4’-epimerase to convert to the desired UDP-GalNAc. After deprotection, the amino function may readily be used for coupling to biomaterials or surfaces to yield a defined multivalent ligand. Generally, glycosidases are not efficient in glycosylation of multivalent acceptors [The upscaled synthesis of the functionalized LacdiNAc disaccharide presented herein is an outstanding example of an elegant, selective, and high-yielding enzymatic synthesis suitable for large-scale biotechnological application. Though the said product may also be prepared by a mutant Tyr284Leu β4-galactosyltransferase from human placenta , glycosycceptors .pNP-GalNAc (maximum approximately 10 mM saturated water solution at 35 °C) was increased, which contributed to a high-yielding GalNAc-ylation. Glycosylation by fungal β-N-acetylhexosaminidases in the presence of organic co-solvents has often proved an advantageous strategy resulting in outstanding synthetic yields [t-butanol is strongly sterically hindered, and therefore its glycosylation is rather improbable, it was observed with fungal α-galactosidase [In this GalNAc-ylation reaction, the presence of organic co-solvent further lowered water activity in the reaction, besides manipulating the enzyme selectivity. Thus, the reaction equilibrium was shifted towards transglycosylation, and, what is more, the concentration of a badly water soluble c yields . Importatosidase . Therefov/v) was used as a mobile phase at a flow rate of 30 μL/min. The sample in methanol was injected into the mobile phase flow through a 2-μL loop under the following conditions: spray voltage 4.6 kV, capillary voltage −25 V, capillary temperature 275 °C. Tube lens voltage was −125 V for the negative ion mode or 150 V for the positive ion mode. The Xcalibur software (Thermo Fisher Scientific) was used for processing the data.The electrospray mass spectrum was measured by the LTQ Orbitrap XL hybrid mass spectrometer . Methanol/water  served as an internal standard; the carbon spectra in D2O were referenced to the acetone signal (δc 30.50). NMR data were analyzed by standard manufacturers’ software TopSpin 3.5. For improving resolution, two-parameter double-exponential Lorentz-Gauss function was applied for 1H. Line broadening (1 Hz) was applied to improve signal-to-noise ratio in 13C. The assignment of individual spin systems in the analyzed compounds was performed by COSY or 1d-TOCSY and transferred to carbons by HSQC. The assignment of quaternary carbons and interconnection of spin systems was done by HMBC experiment.NMR analysis was performed on Bruker AVANCE III 400 and 700 MHz spectrometers  in DTfHex and its three mutant variants  were prepared as described previously [SacI , and electroporated into P. pastoris KM71H competent cells  according to the manufacturer´s protocol . The resulting colonies were tested for the enzyme production as described below. Two colonies with the highest production and purity of the respective enzymes as analyzed by SDS-PAGE and activity measurements were cryopreserved at −80 °C in 15 % (v/v) glycerol.The yeast expression vectors pPICZαA  carrying the genes of WT eviously ,13. The TfHex variants were produced essentially according to the manufacturer´s instructions  [v/v) was added every 24 h to the cultures for inducing the β-N-acetylhexosaminidase expression. On day 5 after the first inoculation, the cultures were purified as described previously [3−) of GE Healthcare, Pittsburgh, PA, USA, equilibrated with 10 mM sodium citrate-phosphate buffer pH 3.5. The elution of the enzyme was done with a linear gradient of 0–1 M NaCl . The concentration of protein was assayed according to Bradford [2HPO4) at pH 5.0.Recombinant ologies) . For a seviously  using caBradford  using PrTfHex variants was measured in end-point assays using the substrate of p-nitrophenyl 2-acetamido-2-deoxy-β-d-glucopyranoside  at a starting concentration of 2 mM. The reaction mixture was incubated in McIlvaine buffer (50 mM citric acid/100 mM Na2HPO4 pH 5.0) for 10 min at 35 °C and 850 rpm. Then, the reaction (50 µL) was stopped by mixing with 0.1 M Na2CO3 (1 mL). The concentration of released p-nitrophenol was determined at 420 nm. One unit of enzymatic activity corresponds to the amount of enzyme releasing 1 µmol of p-nitrophenol per minute under the above conditions. The GalNAcase activity was measured correspondingly, using p-nitrophenyl 2-acetamido-2-deoxy-β-d-galactopyranoside . All data were measured in triplicate.The GlcNAcase of TfHex variants was measured at 35 °C in the same way as above. To cover the wide pH range of 2–10, Britton-Robinson buffer  was used. All data were measured in triplicate.The effect of pH on the activity of pNP-GlcNAc and pNP-GalNAc by TfHex variants were determined in a discontinuous kinetic assay (a continuous assay with pNP-substrates at pH 5 exhibits a very low sensitivity). The reaction mixtures (400 µL) containing substrates at a concentration of 0.01–5.0 mM in McIlvaine buffer pH 5.0 were incubated at 25 °C and 850 rpm for 10–15 min. Aliquots (50 µL) were taken regularly and the reactions were stopped by adding to 100 µL of 1 M Na2CO3. Absorbance (420 nm) was determined by the Spectra Max Plus spectrophotometer . All data were measured in duplicate. The initial reaction velocities were determined by linear regression of respective time points. The kinetic constants  were extracted using GraphPad Prism 7 .Michaelis–Menten parameters of hydrolysis of TfHex mutant variants and molecular dynamics simulations we used the homology model of TfHex described previously [For modeling of eviously . Models eviously  and dockeviously  in minimSubstrate-enzyme complexes were solvated with TIP3 water model with density of 0.997 g/mL, the protonation state of amino acid residues was modeled at pH 6, the protonation of selected active site amino acid residues was corrected manually. Complexes were neutralized by sodium ions and optimized by short minimization with YASARA. Parameters used for molecular dynamics simulation and minimization were similar: periodic boundary condition, YASARA2 force field, Paricle-Mesh Ewald method for long range interactions, NPT (constant number of particles/pressure/temperature) ensemble with Berendsen thermostat and barostat, and AutoSMILES algorithm  were useMolecular dynamics simulations were analyzed with YASARA macros. Selected representative complexes were minimized with OPLS 5 force filed by Protein Preparation Wizard from Schrodinger software and used for GLIDE docking. Binding scores for substrates in the active site of enzymes were calculated by rigid docking with XP scoring method, found to be a superior method for substrate ranging , and aveEcoul) and a term for van der Waals interactions (EvdW). Ebind includes contributions of hydrophobic interactions and hydrogen bonds to the binding, input of π-stacking and π-cation interaction. The penalty term of binding includes an estimate of strain energy (unfavorable contacts), and desolvation of the substrate or protein. It includes an electrostatic term , acceptor 2 , and Tyr470Phe TfHex  were incubated in a mixture of acetonitrile (40 % v/v) and McIlvaine buffer pH 5.0  at 35 °C and 850 rpm. The reaction was monitored by HPLC and TLC . After 2.25 h, another batch of donor 1  was added. After 6.25 h, the reaction was stopped by boiling for 2 min, and upon cooling down to room temperature, the reaction mixture was centrifuged  and loaded onto a Biogel P-2 column  using water as a mobile phase at an elution rate of 7.3 mL/h. Pure acceptor 2 was partially recovered (141 mg). The fractions containing the product were combined and lyophilized; the title compound 3 was obtained as a white solid . HRMS (ESI+): found m/z 648.25211 . For the respective NMR and MS data, see the t-butoxycarbonyl protecting group is deprotected by dissolving compound 3 (20 mM) in 1M HCl and incubating under stirring at 4 °C for 48 h. The reaction mixture is neutralized by adding anex resin in OH− cycle and lyophilized. The deprotected amine 4 ( amine 4  is ratheTfHex under various conditions, namely over a large span of pH and in the presence of water-miscible co-solvents. Thus, apart from a considerable increase in transglycosylation capability and suppression of the undesired side hydrolysis, this point mutation brings a significant stabilization of GalNAcase activity under harsh conditions and the resulting catalyst is highly suitable as a versatile tool for preparative GalNAc-ylations.The present study demonstrates a successful combination of protein and reaction engineering in the large-scale synthesis of a bioactive disaccharide GalNAcβ4GlcNAc, carrying an amino function for multivalent conjugation. This disaccharide is a selective ligand of galectin-3 with a potential application in biomedicine. We have demonstrated the astonishing stabilizing effect of the Tyr470Phe mutation on GalNAcase activity of


Univariate ROC curve analysis showed that the differentially expressed miRNAs could be considered good classifiers of concussion. Further analyses showed significant correlation between these microRNAs and Reaction Time component of the ImPACT concussion assessment tool. In addition, biocomputation analysis predicted the involvement of these microRNAs in important biological processes that might be related to trauma, such as response to hypoxia, cell death, neurogenesis, axon repair and myelination. Ease of access and non-invasiveness of saliva samples make these biomarkers particularly suitable for concussion assessment.Concussion is difficult to diagnose, particularly when symptoms are atypical or late in presenting. An accurate and timely initial assessment is crucial for clinical management. Cerebral spinal fluid (CSF) and blood markers of traumatic brain injury show promising results but their clinical applicability in concussion has significant limitations. In the study, we explored saliva as a new source of biomarkers of concussion. Saliva samples of concussed players were collected after 48–72 h from concussion and analyzed by high-throughput technologies. A discovery group of 10 concussed rugby professional and semiprofessional athletes and 10 non-concussed matched controls was used for the analysis of 92 inflammatory proteins by the Proseek-Multiplex-Inflammation technology. In addition, saliva samples of 6 concussed and 6 non-concussed athletes were used to screen 800 human microRNAs (miRNAs) by the Nanostring Technology. The results were then validated by RT-qPCR in an enlarged cohort  comprising 22 concussed athletes. Results showed, no significant variations of the 65 inflammatory proteins detected in saliva between groups but 5 microRNAs, miR-27b-3p ( There is growing concern that we may be facing a “concussion epidemic” in sport, military and recreational activities in general with significant associated unrecognized morbidity. Concussion, or mTBI  can be dHowever, many studies have demonstrated that individuals who sustain one concussion are potentially more susceptible than others, especially if the new injury occurs before the symptoms from the previous concussion have completely resolved , 2008. IOne of the main challenges faced by medical practitioners is the lack of objective parameters to support the diagnosis of concussion and guide return-to play-decisions following the injury .Currently, the vast majority of concussion assessment tools are indirect measures of the response to trauma. A concussion does not typically cause structural injury to the brain, thus neuroimaging tests such as MRI scan or CT scans are used primarily to rule out a more serious injury, but are not able to exclude the presence of mTBI currently.Over the last few years, the measurement of biomarkers in biofluids has received growing attention. Serum/plasma and CSF are most studied biofluids, but it is possible that some central nervous system (CNS)-derived proteins are eventually excreted into body fluids other than CSF and blood. The presence of the axonal protein Tau in saliva, for example, was demonstrated using mass spectrometry . In addiHowever, the relationship between salivary concentrations of these proteins and processes within the CNS is far from clear and no conclusive data on disease association have been reported so far.Saliva is an important physiologic fluid, containing a highly complex mixture of substances, and is rapidly gaining interest for novel approaches to diagnosis, prognosis and management of patients with either oral or systemic diseases. In addition, it is easily collected and stored, and is ideal for POC devices.In this study, we sought to identify saliva biomarkers from a well-characterized cohort of contact sport-professional and semiprofessional athletes with a view to developing a non-invasive, objective test to support clinical decision-making in sport and military medicine.Study participants were recruited through the Surgical Reconstruction and Microbiology Research Centre (SRMRC), based at Queen Elizabeth Hospital of Birmingham (United Kingdom), as part of the ReCoS (The REpetitive COncussion in Sport). This study was carried out in accordance with the recommendations of the University of Birmingham Research Ethics Committee. The protocol was approved by that same ethical committee together with peer review by the National Institute of Health Research Centre for Surgical, Reconstruction and Microbiological Research Centre (NIHR SRMRC – Ethics Ref. 11-0429AP28). All subjects gave written informed consent in accordance with the Declaration of Helsinki.Male athletes aged 16–65 years, participating in professional and semi-professional Rugby who have been positively diagnosed as having a concussion along with a normal neurological objective examination at assessment, were enrolled in this study. Individuals who require hospital admission after initial assessment for their TBI, presenting intracranial blood, brain tissue injury, or non-TBI related pathologies on initial CT/MR scan, any history of neurodegenerative pathology or history of chronic alcohol or drug abuse were excluded. In addition, age matched controls, who have not received any concussion in the previous 3 months, were recruited.Saliva samples from a total of 20 subjects (discovery group) were analyzed. In particular, saliva samples of 10 concussed and 10 non-concussed athletes were used to screen proteins and 6 concussed and 6 non-concussed athletes for microRNA analysis. Saliva of concussed players was collected 48–72 h after a concussion, certified by the attending enhanced care team in accordance with the current protocol for the relevant sport, was confirmed. Samples from the non-concussed players were collected at rest before the training sessions. The athletes would not have done strenuous exercise since the day before at least.From these data, we calculated the sample size needed for validation in a larger cohort of patients with alpha = 0.05 and power = 0.9. The sample size required was 29 subjects based on the most variant miRNA identified in the discovery group.A second set of samples  was obtained from a total of 22 concussed and 10 non-concussed players. Samples were collected at the same time point.Table 1), neuropsychometric and imaging parameters were collected for each subject including, among others, parameters forming part of the SCAT3 , concussion history, the ImPACT (approved computerized neuropsychometric suite for sport concussion), and the WAIS-IV symbol search.Clinical, demographic .g for 15′ at 4°C. Saliva was then divided into aliquots and to each ml of saliva, 1 ml aprotinin (stock 10 mg/ml), 3 ml Na3VO4 (stock 400 mM) and 10 ml PMSF (stock 10 mg/ml) was added for the analysis of 92 inflammatory proteins. RNase inhibitor (500 units/mL) was added for the analysis of miRNAs.Five ml of saliva were collected in a 50 ml sterile plastic universal container tube kept on ice for no more than 30′. Samples were then centrifuged at 2600 × Samples were stored at -80°C until analysis.Supplementary Table S1) were loaded into the 96.96 dynamic array and the protein expression program activated in the Fluidigm Biomark reader as per Proseek instructions. Further details about detection limits, reproducibility and validations can be found at the Olink webpage1.The Proseek Multiplex Inflammation I  was used to perform the multiplex proximity assay according to the manufacturers protocol . Briefly, 1 μl of human saliva together with 3 μl of mix containing antibodies labeled with corresponding DNA oligonucleotides was incubated over night at 8°C. Following this, 96 μl of extension mix containing proximity extension assay enzymes and PCR reagents were added. Following a 5′ incubation plates were placed on the thermal cycler for 17 cycles of DNA amplification. The 96.96 Dynamic Array IFC  was primed according to the manufacturer’s instructions. In a separate plate, 7.2 μl of detection mix and 2.8 μl of samples were mixed together and from this, 5 μl was loaded into the primed 96.96 Dynamic Array IFC. The specific primer pairs for the 92 inflammatory proteins . Finally, p-values were adjusted for multiple testing through the False Discovery Rate method of correction (FDR).Data were transferred from the Biomark reader to Olink Wizard for GenEx software (Olink). From there, presented as NPX on a linear scale with high NPX corresponding to high protein concentration. Data were checked for normal distribution and ® ND-1000 spectrophotometer . An Agilent 2100 Bioanalyzer  was used to detect the size distribution of total RNA, as well as determine the quality of the RNA. .Total RNA was isolated from 400 μl of saliva by using Qiagen miRNeasy Mini Kit , according to Qiagen Supplementary Protocol for purification of RNA  from serum or plasma . Finally2, using a p-value based on 100 permutations; imputation engine: K-nearest neighbors (10 neighbors); FDR < 0.05.Expression profile of miRNAs from saliva was performed through NanoString technology by using nCounter Human v3 miRNA Expression Assay Kits (NanoString Technologies) in an nCounter FLEX  (NanoString Technologies), according to manufacturer instructions. Profiling was performed on 6 concussed and 6 non-concussed athletes. Three μl (approximately 150 ng) of total RNA were used for sample preparation. Data analysis was performed through nSolver 2.6 software. nCounter “normalized counts” were obtained from the raw counts from each hybridization normalized to the internal positive controls (to account for slight differences in assay efficiencies), and to three most stable reference miRNAs (to account for RNA amount differences), according to nSolver analysis software protocol. MiRNAs used as endogenous controls were selected through GMN method: we computed Pearson correlation between the count means for each lane and the counts of each miRNA, identifying those miRNAs whose expression was closer to the count mean of the cartridge  . StatistHeat map of expression profiles was generated by plotting the fold changes calculated as the ratio between the normalized counts of each sample and the mean of normalized counts of all samples. By using Multi Experiment Viewer (MeV 4.9), we generated the sample clustering through hierarchical clustering approach by selecting Manhattan distance metric.TM). Samples were extracted as described above, retrotranscribed  and RT-qPCR analysis was performed in a Bio-Rad iQ5 Real-time PCR Detection System . Expression fold changes were calculated by the 2-ΔΔCT method by using miR-23a-3p and miR-148b-3p as reference genes.Twenty-one differentially expressed miRNAs were chosen from the arrays as potential candidate biomarkers of concussion. These candidates were used to validate the data in an enlarged cohort of 22 concussed athletes and 10 non-concussed athletes . Saliva was analyzed by single TaqMan assays . We considered for downstream analysis all targets from both tools. Gene ontology enrichment of these targets was computationally analyzed by using FatiGo tool from Babelomics 4 suite 5 and DAVID Bioinformatics Resources 6.86. Statistical over-representation was calculated by using Fisher’s exact test; Benjamini and Hochberg FDR Correction; p ≤ 0.05. The outputs of these analyses were compared and we reported just the matching gene ontologies. The tissue abundance of DE miRNAs was analyzed by screening the Human miRNA tissue atlas7.We retrieved the experimentally validated targets of the Taqman confirmed DE-miRNAs  from TarbaseThe ImPACT test was designed in the early 1990s to assess concussed players of the National Football League .Nowadays, it is one of the most commonly used FDA (food and drug administration) approved neurocognitive tests in sport concussion . The ImPU test) was used to compare the level of miRNAs in the two independent groups. A p-value < 0.05 was accepted as significant.A non-parametric test  analysis was utilized to calculate sensitivity and specificity of each biomarker in diagnosing concussion, expressed as AUC. Multivariate ROC curve was calculated by using MetaboAnalystUsing a Spearman correlation analysis miRNA levels were also correlated with age, ImPACT and WAIS-IV.post hoc test on transformed data.An additional test was applied to compare miRNA expression at different time points, 48–72 h and >120 h, of concussed athletes with non-concussed athletes. The data were checked for normal distribution and transformed to perform parametric tests. Comparisons across groups at each time were performed by the one-way analysis of variance and Tukey’s All statistical analyses were carried on SPSS v.22 (IBM).t-Test was applied for proteins detected and results showed no proteins significantly and differentially expressed between two groups. Volcano plot is represented in Figure 1. A full list of the 65 proteins detected in saliva together with fold changes and standard deviations among groups is also presented in Supplementary Table S2.92 human proteins were analyzed in saliva samples using the Proseek Multiplex Inflammation I panel. All samples met the quality control criteria and 65 of the 92 analyzed proteins were detected in all saliva samples. Among the 800 microRNAs analyzed by nCounter NanoString in saliva of concussed and non-concussed athletes, 21 miRNAs were selected as differentially expressed across the two populations: hsa-let-7c-5p, hsa-let-7i-5p, hsa-miR-15b-5p, hsa-miR-16-5p, hsa-miR-20a-5p+hsa-miR-20b-5p, hsa-miR-24-3p, hsa-miR-27b-3p, hsa-miR-29a-3p, hsa-miR-29c-3p, hsa-miR-30a-5p, hsa-miR-107, hsa-miR-135b-5p, hsa-miR-142-3p, hsa-miR-148a-3p, hsa-miR-181a-5p, hsa-miR-199b-5p, hsa-miR-221-3p, hsa-miR-324-5p, hsa-miR-424-5p e hsa-miR-652-3p. Results showed a significant upregulation for all the miRNAs mentioned above.Figure 2.A heat-map is represented in p = 0.016), let-7i-5p (p = 0.001), miR-142-3p (p = 0.008), miR-107 (p = 0.028), miR-135b-5p (p = 0.017) confirmed the results obtained by Nanostring analysis . AUCs for miR-27b-3p , let-7i-5p , miR-142-3p , miR-107 , miR-135b-5p  are shown in Figure 4. We computed four models of multivariate ROC curves, each one built on a combination of 2, 3, 4, or 5 DE miRNAs, respectively, and compared their AUCs . The “5 miRNA” model showed the highest AUC: 0.836 (CI: 0.669–0.995). However, the comparison among confusion matrixes of univariate curves and “5 miRNA” model curve did not show an improvement of diagnostic performance for multivariate ROC curve, thus confirming let-7i-5p as the best performing biomarker of mTBI (Table 2).In order to validate these findings, we subsequently tested the expression of the 21 selected miRNAs in a separate and independent group, composed of 22 concussed athletes and 10 matched non-concussed athletes. Among these candidate biomarkers for concussion, 5 were significantly upregulated in the validation group. Specifically, miR-27b-3p . By screening the Human miRNA tissue atlas, we found that all validated DE-miRNAs, with exclusion of miR-142-3p, were expressed in almost all body tissues, but more abundant in brain .The different distribution of these miRNAs in saliva of concussed athletes is most likely a systemic consequence of the different physiopathology of this typology of trauma. In order to evaluate the biological functions of DE-miRNAs, we computationally searched their validated or predicted targets. Gene ontologies and pathway associations of miRNA targets were analyzed by FatiGo and DAVID. This analysis showed that they could be involved in important biological processes related to trauma   and miR-27b-3p . In addition an inverse correlation was detected between the level of miR-135b-5p and the number of concussions  .A summary of the ImPACT and WAIS data obtained from the concussed and non-concussed groups is illustrated in U, p = 0.54 and 0.204, respectively) between the concussed and non-concussed group.No statistically significant difference was seen in the ImPACT cognitive efficiency index , or WAIS symbol search score , let-7i-5p (p = 0.014), miR-142-3p (p = 0.028), miR-107 (p = 0.015), miR-135b-5p (p = 0.042).In order to assess the progress or decline of the selected miRNAs at later time points from concussion, we subsequently tested the expression of the 5 selected miRNAs in a separate and independent group, comprising 12 concussed athletes. Expression changes were compared with early concussed athletes (48–72 h) and non-concussed athletes. Results showed in p = 0.036) between concussed athletes early time point (48–72 h) and concussed athletes later time point (>120 h). No others significant results were found across the groups.An additional significant result was found in the fold change of let-7i-5p , showed that these miRs are not able to discriminate concussed from non-concussed athletes.Several limitations of this study must be considered.In the first instance, the lack of miRNA validation. Only 5 of the 21 miRs selected in the discovery study, which means <25% of candidates, were confirmed in the validation cohort of the 22 athletes analyzed. This can be due to several factors; the sample size for example, although partially justified with the power analysis, is smaller than existing microRNA studies . SecondlCurrently, the majority of the studies on miRNA biomarkers, reports the comparison between miRNAs identified in patients and in healthy controls without testing other factors that impact the miRNA abundance. For this reason, the evaluation of miRNA biomarkers in terms of stability and variability in a population of individuals without known diseases, remains one of the main challenges. Thus, variable markers can only be used if there are substantial differences in the signal between affected and unaffected individuals.Another limitation of the study is that long-term outcome including return to play data and chronic symptomatology in these patients was not collected. Finally, we do not know how soon these biomarkers became upregulated in saliva after concussion.Further research, including an earlier and detailed time of collection together with an enlarged group of study and a comparison with internal controls such as within the same subjects before and after concussion, or with orthopedic injury population and an additional study showing microRNA variation after exercise, is needed in order to aid the diagnosis of concussion in the treatment room, clinic and possibly pitch-side.However, the most important finding showed in this paper, is that saliva miRNA outperforms saliva protein for identifying mTBI and provides support for the idea that peripheral microRNA patterns hold promise where years of protein biomarker work have fallen.VD conception and design of the study, acquisition and analysis of data, and drafting the manuscript or figures. EP, MR, CB, DD, MF, and CD contributed to the data acquisition and analysis. AL, MP, MG, DH, VS, AKB, and AB conception and design of the study and editing the manuscript or figures. All authors approved the final version.The University of Birmingham has intellectual property associated with miRNA listed in this manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Immune checkpoint inhibitors are effective in some cases of lung adenocarcinoma (LUAD). Whole-exome sequencing has revealed that the tumour mutation burden (TMB) is associated with clinical benefits among patients from immune checkpoint inhibitors. Several commercial mutation panels have been developed for estimating the TMB regardless of the cancer type. However, different cancer types have different mutational landscapes; hence, this study aimed to develop a small cancer-type-specific mutation panel for high-accuracy estimation of the TMB of LUAD patients.We developed a small cancer-type-specific mutation panel based on coding sequences (CDSs) rather than genes, for LUAD patients. Using somatic CDSs mutation data from 486 LUAD patients in The Cancer Genome Atlas (TCGA) database, we pre-selected a set of CDSs with mutation states significantly correlated with the TMB, from which we selected a CDS mutation panel with a panel-score most significantly correlated with the TMB, using a genetic algorithm.p = 0.0018, HR = 3.35, 95% CI 1.51–7.42; log-rank p = 0.0020, HR = 5.06, 95% CI 1.63–15.69). This small panel better predicted the efficacy of immunotherapy than current commercial mutation panels.A mutation panel containing 106 CDSs of 100 genes with only 0.34 Mb was developed, whose length was much shorter than current commercial mutation panels of 0.80–0.92 Mb. The correlation of this panel with the TMB was validated in two independent LUAD datasets with progression-free survival data for patients treated with nivolumab plus ipilimumab and pembrolizumab immunotherapies, respectively. In both test datasets, survival analyses revealed that patients with a high TMB predicted via the 106-CDS mutation panel with a cut-point of 6.20 mutations per megabase, median panel score in the training dataset, had a significantly longer progression-free survival than those with a low predicted TMB (log-rank The small-CDS mutation panel of only 0.34 Mb is superior to current commercial mutation panels and can better predict the efficacy of immunotherapy for LUAD patients, and its low cost and time-intensiveness make it more suitable for clinical applications. Lung adenocarcinoma (LUAD) is the most common type of non-small cell lung cancer (NSCLC), accounting for approximately 80–90% cases of lung cancer . CurrentPreviously, PD-L1 protein expression in NSCLC patients has been approved as an auxiliary predictive marker for certain PD-1/PD-L1 inhibitors including pembrolizumab . HoweverSeveral previous studies have reported a high tumour mutation burden (TMB), determined through whole-exome sequencing (WES), indicating that patients are more likely to harbour neoantigens, can predict the sensitivity to immunotherapies , 14. ForIn this study, based on the coding sequences (CDSs) with a high frequency of mutation in LUAD, we developed a CDS mutation panel to estimate the TMB of LUAD samples. Thereafter, we determined the correlation of CDSs in the mutation panel with the TMB in two independent datasets. From two datasets (Matthew and Rizvi), we used data on progression-free survival (PFS) of advanced LUAD patients treated with immune checkpoint inhibitors to estimate the performance of the CDS mutation panel for predicting the efficacy of immunotherapy. Furthermore, the CDS mutation panel was compared with two commercial mutation panels (324-gene and 341-gene panels) and a LUAD-specific mutation panel (24-gene panel).https://portal.gdc.cancer.gov/), comprising 486 LUAD samples with paired mRNA expression data. For validation, we obtained two independent somatic mutation datasets with PFS data for patients treated with immune checkpoint inhibitors, including 59 LUAD samples reported by Matthew et al. [The three LUAD somatic mutation datasets Table  were usehttps://www.gencodegenes.org/human/releases.html).Whole-exome sequencing was previously performed for these TCGA data with tumour tissues and matched normal tissue or blood, which were used to filter out germline mutations and screen somatic mutations ; the detm*n CDSs mutation matrix, where m represents the number of CDSs in genes and n represents the number of samples. TMB was estimated as  * 106.First, from TCGA LUAD somatic mutation data, we extracted mutations in the CDSs using the human reference genome annotated file (GRCh38), and selected non-synonymous mutations to construct an Thereafter, Spearman’s rank correlation analysis was performed to estimate the correlation of the CDSs mutation state with the TMB. Herein, we restricted the analysis to the CDSs mutated in more than 5% cancer samples , 30 to f2) was estimated via linear regression analysis [n is the number of CDSs in the panel, l is the length of the panel, and i-th CDS; Finally, the genetic algorithm (GA package) was used to generate a final CDS panel from among candidate CDSs, whose panel-score was most correlated with TMB. The genetic algorithm was implemented with a population size of 5000 and a crossover fraction of 0.9; it was terminated if the optimization objective of the best subset was not improved in 100 generations. Details regarding the genetic algorithm are shown in Additional file analysis . Here, tAs no clinical data regarding immunotherapy were available for patients in TCGA, we could not determine the optimal cut-point for our CDS panel for predicting the efficacy of immunotherapy. Therefore, we set the cut-point of our CDS panel at a median panel score in TCGA.PFS was defined as the period during and after the treatment of a disease, wherein a patient lives with the disease but it is not exacerbated. The survival curve was estimated using the Kaplan–Meier method and compared using the log-rank test  . The uniFunctional pathways for enrichment analysis were downloaded from Gene Ontology (GO) in November 2018. First, we performed Student’s t-test with a 5% FDR control to select differentially expressed genes (DE genes) between the high-TMB and low-TMB groups classified by the CDS panel. Here, 17,680 genes were used for differential expression analysis. Thereafter, the hypergeometric distribution model was used to determine whether the number of DE genes observed in a functional term was significantly greater than that expected through random chance.http://www.r-project.org/). Significance was defined as p < 0.05 or FDR < 0.05 for multiple testing.All statistical analyses were performed by using R software packages version 3.4.2 . After selecting non-synonymous mutations, a mutation matrix comprising 82,574 CDSs and 486 patients was generated. Thereafter, using Spearman’s rank correlation analysis, with a 5% FDR control, 219 CDSs were significantly correlated with the TMB of the LUAD samples derived from TCGA data. Using the genetic algorithm “”, we extanalysis . The tec8. After p = 0.0055; sex: p = 0.0442, Additional file 2) of the 106-CDS panel with the recalculated TMB also approached 0.94  between the panel-score of the 106-CDS and the TMB was 0.82  between panel-score and the TMB was 0.73  with a cut-point of 141 , containIn TCGA dataset, using the 106-CDS panel with a cut-point of 6.20 mut/Mb, 220 and 266 samples were divided into high- and low-TMB groups, respectively. We found that 7181 genes were differentially expressed between the two groups  of the predicted low-TMB patients. Similarly, the 1-year PFS rate of the predicted high-TMB patients was 0.61, being markedly higher than the 1-year PFS rate 0.13) of the predicted low-TMB patients after pembrolizumab treatment. However, we considered that the cut-point of the 106-CDS panel, which was set at a median panel score in training dataset, may not be the optimal threshold for predicting the efficacy of various immunotherapy drugs. In order to assess the effect of specific cut-points for predicting the efficacy of immunotherapy, we additionally set the cut-points of our CDS panel at upper tertiles (9.17 mut/Mb) and quartiles (12.13 mut/Mb) of panel scores in training dataset, respectively, and estimated in the two test datasets. The univariate survival analyses revealed that the 106-CDS panel with the cut-point of the upper quartiles (12.13 mut/Mb) had the optimal predictive performance (log-rank  of the pThe larger the number of genes included in the mutation panel, the higher the expected correlation with the TMB. Our results show that although the number of genes in the 106-CDS panel is twofold less than that of the 324-gene  and 341-Functional annotation revealed that several genes including TP53 , AMER1 , and TEXThe CDS mutation panel spanning only 0.34 Mb can effectively predict the efficacy of immunotherapy for LUAD patients through accurate estimation of the TMB. This small panel is preferable for clinical samples because of its low cost and time consumption.Additional file 1. The genetic algorithm for searching a CDS panel.Additional file 2: Table S1. Description of the 106-CDS panel.Additional file 3: Table S2. Technical details of TMB evaluation.Additional file 4: Table S3. The correlation of the clinical factors and tumour cell proportions with the tumour mutation burden.Additional file 5: Table S4. List of differently expressed genes between high-TMB and low-TMB groups predicted via the 106-CDS panel.Additional file 6: Table S5. The functional pathways enriched with differently expressed genes.Additional file 7: Figure S1.  Performance of 106-CDS panel with the other cut-points for predicting the efficacy of immunotherapy.


The collectivism can be divided into two forms: relational collectivism and group collectivism. According to the cognitive representation of self, relational collectivism emphasizes the relational-self and group collectivism privileges the collective-self. However, it remains uncertain whether there is a difference between relational-self and collective-self under Chinese collectivism cultural. To address the above issue, the present study examined the neural representation of relational-self and collective-self during trait judgment tasks using functional magnetic resonance imaging (fMRI). The results showed that relational-self-reference compared with collective-self-reference generated stronger medial prefrontal cortex (MPFC) activity, indicating relational-self was more closeness and important in the self-concept than collective-self under East Asian cultural background. Relational-self and collective-self are unequally represented in the MPFC, providing direct neural evidence that the collectivism in China can be divided into relational collectivism and group collectivism. There is considerable evidence that people have three cognitive representations of the self: the individual-self , the relational-self (cognitions related to relationships with close others), and the collective-self (cognitions related to membership groups) . A serieOne of the central claims of cultural psychology is that an individual’s representations of self are influenced by individual’s culture of origin. The model of cultural difference in self-construal claims that East Asian cultures emphasize fundamental social connection with others and social group and encourage an interdependent view of a self. The interdependent self-construal model corresponds to the collectivism cultural descriptor . FurtherThe neural basis of the self has been recognized as one of the most prominent problems in neuroscience . ResearcSimilar to the individual-self, the information of relational and collective selves has high emotional significance, reward value, and/or more familiarity compared with other information . EspeciaLike the relational-self, the collective-reference effect was also confirmed in Chinese subjects. The goal of this study was to investigate the different neural mechanism of relational-self and collective-self in Chinese people. To achieve the above issue, we adopted a trait judgment task to assess the representation of relational-self and collective-self . Mother Accordingly, the present study was designed to examine the neural mechanism of collective-self and determined whether the brain activation of relational-self would be different compared with collective-self. If the brain activation patterns in relational-self-processing condition were different from collective-self-processing, this may indicate that the collectivism in China can be divided into relational collectivism and group collectivism. Based on these analyses, we hypothesized that the relation-self may activate stronger MPFC than collective-self during the trait-judgment processing. Understanding the complexities of relational collectivism and group collectivism and their interrelationships may prove to be critical to managing social change.2 = 0.35), moderated power (1-β = 0.80), and a within factors repeated measures F-test, approximately 20 participants were required. Data collection ceased at the end of the academic term in which this minimum number was reached.Prior to data collection, a power analysis was performed to determine the necessary number of subjects. Assuming a moderate effect size   were recruited from Southwest University of China. Participants were all right handed and reported no abnormal neurological or psychiatric history. All of them had normal or corrected-to-normal visual acuity. Informed consent was obtained in accordance with procedures and protocols that were approved by the human subjects review board of Southwest University in China. All methods were carried out in accordance with the approved guidelines.∗). Each null session lasted for 44 s. The order of the judgment tasks was randomly for each participant and the bottoms were counterbalanced between subjects.Participants were scanned during performing trait judgment tasks, and six functional runs were carried out for each subject. The stimuli were presented through an LCD projector onto a rear projection screen at the head end of the bore. The screen was viewed with an angled mirror positioned on the head coil. Three functional scans involved relational-self and the others involved collective-self. Each of the functional scans consisted of three judgment blocks. Three judgment tasks were conducted in each relational-self scan requiring subjects to judge if an adjective was proper to describe the mother, Xun Lu (a well-known Chinese writer) or to judge the valence of the words (positive or negative) Figure . In addiEach trial in the judgment tasks consisted of a “cue” word  above a trait adjective presented for 2000 ms at the center of the screen. The trait adjective then disappeared while the ‘cue’ word stayed on the screen for 1000 ms, during which time participants made their responses. After each trail, the response was followed by a blank which was presented for 1 s before the next trial. Each of the Chinese characters in the cue word and trait words subtended a visual angle of 0.34° × 0.45° (width × height) and 0.85° × 1.14°, respectively at a viewing distance of 80 cm. Each symbol used in the null session subtended 0.23° × 0.23°  or 0.56° × 0.56° (large ones). All words and symbols were white on a black background.A total of 240 unique adjectives consisting of two Chinese characters were selected from established personality trail adjective pools  sequence was used to acquire high-resolution T1-weighted anatomical images.Function imaging data was acquired using a Siemens TRIO 3T MRI scanner in the Key Laboratory of Cognition and Personality at the Southwest University in China. Participants lay supine with their heads snugly fixed with foam pads to minimize head movement and were instructed to keep still. A total of 150 BOLD Images were obtained using an Echo Planar Imaging (EPI) sequence with the following parameters: slices = 32; repetition time (TR) = 2 s; echo time = 30 ms; field of view (FOV) = 220 mm × 220 mm; flip angle = 90°; voxel size = 3.4 mm × 3.4 mm × 3 mm; thickness = 3 mm; matrix = 641 was used for data preprocessing analysis. Functional images were corrected for the acquisition delay between slices using the middle slice as the reference frame and further realigned to the first volume to correct for head movement. Participants with head motion exceeding ± 3.0 mm of translation or ± 3.0° of rotation were excluded from the dataset. Functional images were then normalized to EPI templates based on the Montreal Neurological Institute (MNI) space (resampling voxel size was 3 mm × 3 mm × 3 mm). The resulting normalized data were then spatially smoothed (8 mm FWHM Gaussian kernel) to increase signal to noise ratio.DPARSF 2.3p < 0.05 . In the first-level analysis, the onsets and durations of each session were modeled using a General Linear Model (GLM) according to stimulus conditions. The movement correction parameters were added as covariates of no interest. All six conditions  were included in the model. The contrast between trait judgments (mother/famous/Chinese/American) and matched valence judgments was defined in each subject. These individual contrast images were submitted to a second-level random-effect analysis. The significance threshold was set at 2. Contrast values were extracted from the ROIs by subtracting the coefficient estimates of the valence judgments condition from those of the trait judgment conditions. The fMRI signals were subjected to a repeated measure analysis of variance (ANOVA) with factors being Self  and Information type (self-relevant vs. non-self-relevant). Then the fMRI signals in the non-self-relevant condition were also subtracted from the self-relevant condition to index the relational-self and collective-self reference effects, respectively. A paired t-test was then conducted on the differential fMRI signals.A regions of-interest (ROI) was conducted to explore the representation difference of relational-self and collective-self in MPFC. ROI was defined as spheres centered at the peak voxels of MPFC activation in the contrast between relational-self and collective-self reference conditions, with radius of 6 mm using MarsBaRM = 5.29, SD = 0.43 vs. M = 5.12, SD = 0.42, t(29) = 2.35, p < 0.05]. These results suggest collectivist cultural orientation among out Chinese sample. On the 7-point Likert scale of closeness between self and mother , the participants’ rating scores ranged from 2 to 7 , suggesting the closeness between participants and their mothers was above the middle level, t(29) = 3.64, p = 0.001.Results from the SCS showed that the participants were tended to be interdependent, self-construal compared with independent self-construal [F = 0.52, p > 0.05, and no significant interaction of self-construal and information type, F = 0.42, p > 0.05. However, the main effect of information type was significant, F = 6.64, p < 0.01, η2 = 0.32, observed power = 0.88. Bonferroni-corrected post hoc analyses showed that response latencies were significantly faster for valence-processing than for non-self-relevant information-processing, p < 0.01. However, the comparison of response times between self- and non-self-relevant information processing did not reach significant.Figure To identify brain areas involved in the different encoding process, the whole brain analysis of mother-, famous-, Chinese-, and American-judgments were first contrasted with matched valence-judgment Table . The rest-test. The results found that ventral medial prefrontal cortox (vMPFC) showed increased activity linked to mother-judgments processing than famous-judgments, whereas the activations during Chinese-judgments and American-judgments processing were comparable  than collective-self (10 contiguous voxels at p < 0.001 uncorrected).To assess the difference between relational-self reference and collective-self reference effects, we defined the neural substrates of the self-reference effect as increased neural activities associated with self-relevance- than non-self-relevance-judgments. The contrast between mother- and famous-judgments was used to index the relational-self reference effect, and the contrast between Chinese- and American- judgments to index the collective-self reference effect. The results of paired F = 6.83, p = 0.01, η2 = 0.17, observed power = 0.71. The interaction of self and information type was also significant, F = 13.06, p = 0.001, η2 = 0.28, observed power = 0.94. Simple effect analysis revealed that signals in MPFC was greater to mother- than famous- and Chinese-judgment, both p = 0.001, but did not differ between Chinese- and American-judgments, p = 0.41  = 3.27, p < 0.01 relative to the matched valence judgments condition. 2 × 2 repeated ANOVA was conducted on the differential ROIs of the contrast values. The results confirmed that the main effect of self did not reach significant. However the main effect of Information Type was significant, 1 Figure . Then a To examine whether the MPFC activity associated with the relational- and collective-self can predict subjective evaluations of one’s self-construal and intimacy with mother, we calculated correlations between subjective rating scores and MPFC activity from the ROI defined in the contrast in association with relational- and collective-self reference. No significant correlation was found.Using fMRI, we examined the neural mechanism during the collective and relational-self–reference processing. The results showed greater activation in the MPFC during mother- and Chinese-judgment processing compared to valence processing. Furthermore, there is a stronger activity of MPFC during mother-judgment compared with Chinese-judgment. Moreover, after subtracting the non-self-relevant information judgments, the relational-self-reference showed greater MPFC activation than collective-self. These results provided first evidence for greater brain activation in MPFC of relational-self compared with collective-self, suggesting that relational-self was more closeness and important in the self-concept than collective-self under East Asian cultural background.The aim of the current study was to compare the hierarchical differences during cognitive processing between relational-self and collective-self. ROI analysis revealed that Chinese-judgments processing exhibited smaller activation of MPFC than mother-judgments processing. Consistent with previous motivation researches , the resMoreover, the activation of the relational- and collective-self was more obvious on the left hemisphere, whereas considerable research indicated that the right hemisphere played a crucial role in self-referential processing , 2009. HThe fMRI results showed the MPFC was activated both during mother-, and Chinese-judgments processing compared with valence-judgment processing. A large body of research on the self-reference has established that MPFC is associated with self-related processing and coded self-related information, including self-reflection on personality traits, self- face recognition, and self-related information retrieval . TherefoConsistent with previous researches, mother-judgment activated stronger vMPFC than famous-judgment processing. Studies of self-reference processing provided evidence that vMPFC was primarily responsible for tagging self-related information, and involving in the automated social cognitive processing of experienced areas . CompareOne limitation of our study is that our sample consisted solely of Chinese participants with interdependent self-construal. Given that the overlapping neural representation between the self and mother is stronger in Chinese interdependent self than in Westerners independent self , it is pThe current study compared the processes engage and neural mechanism during the relational-self- and collective-self-reference. The direct comparison of relational- and collective-self enriched the study of the cognitive hierarchy of self in Chinese brain. The MPFC activation was stronger during relational-self compared with collective-self reference, indicating that the processing of relational-self involves more attentional resources to identify and evaluate relevant memories and emotional information, and confirming that the Chinese people emphasis more relational collectivism than group collectivism. In addition, the left lateralization for relational- and collective-self indicated that the influence of social situation on self was mainly represented in the left brain. The different brain activation of relational- and collective-self confirmed that the collectivism in China can be divided into relational collectivism and group collectivism, and different social connection such as interpersonal relationship and depersonalized social category have distinct effect on one’s self.http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided that appropriate credit is attributed to the original author(s) and the source provides a link to the Creative Commons license and indicates whether changes were made.This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study.YZ, ZX, and HC conceived and designed the experiments. YZ and ZX performed the data analysis and wrote the manuscript. LW and HC reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.